Research at the DNA replication group aims at understanding how information from the cell cycle machinery leads to the initiation of DNA replication. Recent studies yielded abundant information about signaling pathways that prevent normal cells from proliferating in response to developmental cues or to damage sensor signals. Cancer results from the inability to respond to these signals, leading to DNA replication and mitosis under potentially genotoxic conditions. Such replication may promote genetic instability by inducing mutations and chromosomal aberrations. Although DNA replication is the ultimate endpoint of cell cycle signaling pathways, very little is known about how signals from damage sensors and cell cycle regulators reach the DNA duplex. Our studies probe into this missing link through the analysis of molecular factors that determine the site and the timing of DNA replication. Our current research plan focuses on two aspects of initiation: DNA sequences that dictate replication initiation sites, and determinants of replication timing. Replication sites. Our previous studies focused on sequence elements that determine the sites from which DNA replication initiates in mammalian chromosomes, termed "replicators". We had previously developed an assay to measure replicator function in mammalian cells using site-specific recombination and PCR-based initiation analysis. Using this assay, we identified replicator activity in the human beta-globin locus (Aladjem, et al., 1998. Science 281(5379): 1005-1009.). During the last two years, we have extended the genetic dissection of the locus and identified several sequence elements within the globin replication initiation region, organized in two independent replicators, which cooperate in initiation of DNA replication. We have determined that several sequence motifs that had been implicated in initiation of DNA replication, such as "hairpin" structures, are not essential for initiation. We are currently focusing on a newly identified sequence motif, which is conserved between various human globin haplotypes, that seems to play a role in the initiation of DNA replication. We have completed an analysis of the replication initiation profile in the murine beta-globin locus. Using a combined biochemical and genetic approach, we has shown that replication initiates from multiple sites within the murine human b-globin locus. While the initiation sites were not conserved, replication timing was similar in murine and human cells and subject to developmental control. Replication timing. Since previous studies have indicated that the timing of replication in the beta-globin region is regulated by tissue-specific chromatin remodeling factors. We are using this locus as a model to understand the effect of alterations in chromatin packaging and gene expression on the initiation of DNA replication. To identify the determinants of replication timing, we have characterized a series of cell lines harboring silenced and active globin transgenes. She recently found that replication initiates early during S phase in the "expressed" transgene, while in the "silenced" transgene, replication initiates late. Early replication correlates with histone acetylation in the vicinity of the transgene. During silencing, late replication precedes histone deacetylation and transcription deactivation. These data contrast, but can be reconciled with, previous observations suggesting that replication timing domains tend to be very large chromosomal regions and that transition between early and late replication is gradual. Our observations imply that an insertion of a relatively small sequence can alter replication timing, and suggest a way to probe into the nature of the cis-acting elements that regulate the timing of DNA replication. Cell cycle control on DNA replication - bioinformatic tools. In the scientific literature, various studies have identified a plethora of candidate proteins that may be involved in regulating DNA replication during the normal course of the cell cycle and under conditions where DNA replication is perturbed. To gain better insight into which of these proteins might be the best candidates for replication initiators in mammalian cells, it is necessary to categorize the knowledge accumulated from various systems in an organized manner. We have generated a molecular interaction map illustrating the initiation of DNA replication. A concise version of such a map is currently in press (Encyclopedia of the human genome) and an interactive version is being developed on the LMP web site. This work is a part of a collaboration with Dr. Kurt Kohn, Yves Popmmier and John Weinstein.